Erratum in

J Exp Med 1998 Aug 3;188(3):following 614.

Abstract

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

(A) Downregulation of class I MHC in hCMV-infected HFFs. AD169 or mock-infected HFFs were stained with anti–class I (DX17) or control Ig (cIg), followed by PE-conjugated goat anti–mouse Ig. HFFs were infected with AD169 at an MOI of 3, and cells were stained at 24 h after infection. (B) NK cell cytotoxicity assay against hCMV or mock-infected HFF cells. hCMV or mock-infected HFFs were used as targets in a 4-h 51Cr–release assay. HFFs were infected at an MOI of 3 and used at 48 h after infection. Mock-infected, white squares; AD169, black diamonds. (C) Blocking class I MHC, KIR, or CD94 does not induce NK cell killing of uninfected HFFs. Normal HFFs were incubated in the presence of NK cells and with cIg or a mixture of mAb against class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a mixture of anti-KIR mAbs (DX27, DX30, DX31, and HP-3E4) and anti-CD94 mAb (DX22), each at 20 μg/ml. cIg, black bars; anti-KIR/CD94, gray bars; anti–class I, hatched bars. (D) AD169 or mock-infected HFFs were incubated in the presence of NK cells with cIg or mAb directed at class I MHC (DX15, DX16, and DX17 at 20 μg/ml) or a cocktail of mAbs against KIR and CD94. Cytotoxicity assays were performed at E/T ratios of 5:1. As controls, 721.221 class I HLA transfectants expressing HLA-B*0702 or HLA-Cw*0702 were analyzed, using NK clones expressing the relevant CD94/NKG2A or KIR, respectively. cIg, white bars; anti-KIR/CD94, black bars; anti–class I, hatched bars.

(A) Downregulation of class I MHC after Δ18 and AD169 hCMV infection of HFFs. HFFs were infected with Δ18 or AD169 at an MOI of 5. At 48 h after infection cells were stained with anti–class I MHC or cIg, followed by PE-conjugated goat anti–mouse second step. (B) Expression of UL18 in AD169 but not Δ18 cell lysates. Lysates were prepared from HFF-infected with Δ18 or AD169 at 48 h after infection and blotted with anti-UL18 or anti-hCMV IE mAb, followed by horseradish peroxidase–conjugated second step. (C) AD169-infected HFFs were lysed more efficiently than were Δ18-infected HFFs. HFFs were infected with AD169 or Δ18 at an MOI of 5, and used as targets at 48 h after infection in 4-h 51Cr–release assays. Assays were performed at an E/T ratio of 20:1, 10:1, and 1:1. Standard deviation between triplicates was <10%. Δ18, black circles; AD169, black diamonds; mock-infected, black squares. (D) Surface UL18 expression on 293EBV transfectants. 293EBV cells were transfected with pREP10 UL18. Cells were cultured for 48 h before use in cytotoxicity assays. Histograms of sorted UL18-positive cells. Cells were stained with anti-UL18 mAb 10C7 followed by PE-conjugated goat anti–mouse Ig. (E) 293EBV transfectant expressing UL18 were lysed at an enhanced level compared with parental controls. UL18-transfected 293EBV cells were used as targets in NK cell cytotoxicity assays. Experiments were performed at an E:T ratio of 5:1. All transfectants other than vector only were positively sorted using mAb directed against the protein encoded by the transfected cDNA. Cells were cultured for 48 h and used in 4-h cytotoxicity assays as described above.